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doi: 10.1242/10.1242/jcs.00292

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{gamma}-Secretase activity requires the presenilin-dependent trafficking of nicastrin through the Golgi apparatus but not its complex glycosylation

An Herreman1, Geert Van Gassen1, Mustapha Bentahir1, Omar Nyabi1, Katleen Craessaerts1, Ulrike Mueller2, Wim Annaert1,* and Bart De Strooper1,*,{ddagger}

1 Laboratory for Neuronal Cell Biology, Center for Human Genetics, Gasthuisberg/KULeuven and Flanders Interuniversity Institute for Biotechnology (VIB), Herestraat 49, 3000 Leuven, Belgium
2 Max-Planck-Institute for Brain Research, Department of Neurochemistry, D-60584 Frankfurt, Germany



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  		Fig. 1. Glycosylation modifications of nicastrin and effect of PS deficiencies. (A) Western blot analysis of total cell extracts from wild-type, PS1–/–, PS2–/– and PS1–/–PS2–/– MEFs using an antibody raised to the C-terminus of nicastrin (B59.2). Cell extracts were subject to digestion with endoH (H) and N-glycosidase F (F); –20C and 37C represent control samples (no addition of enzyme) kept at –20°C and 37°C. Note the partial endoH resistance of mature nicastrin (NCTm) in wild-type and in PS2–/– MEFs, and in PS1–/– MEFs to a lesser extent (overexposed part of the blot below). In PS1–/–PS2–/– MEFs, only endoglycosidase-sensitive immature nicastrin (NCTi) is observed (see also overexposed inset). (B) Lectin binding to nicastrin. Nicastrin was immunoprecipitated from total cell extracts of wild-type MEFs and blotted using the indicated lectins. The immature glycosylated nicastrin reacted strongly with Galanthus nivalis agglutinin (GNA), indicating the presence of terminally linked mannose residues. NCTm also reacted weakly with this lectin. Furthermore, NCTm reacted with Maackia amurensis agglutinin (MAA) and Datura stramonium agglutinin (DSA), indicating the presence of sialic acid residues and complex and hybrid N-glycan structures, respectively. No reaction was observed with peanut agglutinin, specific for O-glycan modifications. Control glycoproteins demonstrated the specificity of the lectins. (C) Western blot analysis of total cell extracts from wild-type and PS1–/– neurons. C, control (no enzyme); H, endoH digestion; F, N-glycosidase F digestion; N, neuraminidase digestion; O, O-glycosidase digestion. Notice the partial sensitivity to endoH and the complete sensitivity to N-glycosidase F of neuronal nicastrin. A small shift in mobility is also observed after neuraminidase digestion. (D) Western blot analysis of total cell extracts from wild-type and APP–/– MEFs. C, control (no enzyme); H, endoH digestion; F, N-glycosidase F digestion. No differences between wild-type and APP–/– MEFs could be observed.

 


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  		Fig. 6. Pulse chase of nicastrin in wild-type and PS-deficient cells. (A) Wild-type (WT) and PS1–/–PS2–/– (KO) MEFs were metabolically labeled for 15 minutes and chased for the indicated time periods. Nicastrin was immunoprecipitated as described. In WT MEFs, the amount of immature nicastrin decreases in time, while mature nicastrin slowly increases. Note that mature nicastrin is extremely stable. In PS1–/–PS2–/– MEFs, no mature nicastrin is observed. Immature nicastrin degrades slowly. (B) WT and PS1–/–PS2–/–MEFs were metabolically labeled for 15 minutes and chased for the indicated time periods. APP was immunoprecipitated as described. Note the rapid degradation of both mature (APPm) and immature APP (APPi). (C) Graph showing immature/mature nicastrin as a function of time.

 


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  		Fig. 2. Binding of mature nicastrin to PS. (A) PS1 NTF, PS1 CTF and nicastrin were immunoprecipitated from total cell extracts of wild-type MEFs and blotted with B59.2 ({alpha}-nicastrin). As a control, pre-immune serum (PI) was used for immunoprecipitation. Both PS1 NTFs and CTFs interact preferentially with the mature form of nicastrin (NCTm). (B) Wild-type MEFs were treated for 24 or 48 hours with MNJ (0.2 mg/ml). This treatment inhibits mannosidase I and therefore the full maturation of the glycosylation of nicastrin. Only endoH-sensitive nicastrin is found. (C) Wild-type MEFs were treated for 48 hours with MNJ (0.2 mg/ml). PS1 NTF and NCT were immunoprecipitated from total cell extracts and blotted with B59.2 ({alpha}-NCT). As a control, PI was used for immunoprecipitation. Note that deglycosylation of nicastrin does not inhibit interaction with PS1.

 


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  		Fig. 3. Surface expression of nicastrin is disturbed in PS-deficient cells. Wild-type (WT) and PS1–/–PS2–/– MEFs (KO) were surface biotinylated. Biotinylated proteins were precipitated using streptavidin beads and immunoblotted with B59.2 ({alpha}-nicastrin). Western blot analysis of total cell extracts is shown as a control. Note that only in WT MEFs is mature nicastrin labeled.

 


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  		Fig. 4. PS deficiency affects the subcellular localization of endogenous nicastrin. Wild-type (top panel) and PS-deficient (bottom panel) MEFs were grown on coverslips at 37°C, then fixed and processed for double immunofluorescence staining using antibodies against the ER chaperone BIP (left) and nicastrin (middle). Detection was carried out with Alexa-488 and -555 conjugated secondary antibodies, respectively. In wild-type MEFs, little overlap is seen between BIP and nicastrin. By contrast, PS deficiency clearly results in increased localization of nicastrin in the ER (compare merged pictures and insets). Bar, 10 µm.

 


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  		Fig. 5. Endogenous nicastrin and PS1. PS-deficient MEFs stably expressing human PS1 were grown on coverslips at 37°C, then fixed and triple labeled using antibodies against calnexin, human PS1 and nicastrin. Alexa-488, -647 and -555 conjugated secondary antibodies were used to detect the respective primary antibodies. Individual stainings and the merged picture of the whole cell (top panels), as well as detailed areas (bottom panels and insets), are shown. Both human PS1 and endogenous nicastrin only partially colocalize with the ER marker calnexin. Whereas human PS1 and nicastrin can be clearly seen together in discrete structures, in many cases no co-localization is observed (pink versus blue color in merged pictures and insets). Bar, 10 µm.

 


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  		Fig. 7. {gamma}-Secretase activity is not influenced by the glycosylation status of nicastrin. (A) Cell-free {gamma}-secretase assay using membranes from wild-type (WT) MEFs treated with MNJ. C–, negative control (substrate only); C+, positive control (solubilized {gamma}-secretase from Hela cells); –s: solubilized {gamma}-secretase from WT MEFs without substrate added. +s: substrate added. (B) Western blot analysis of the membranes used in panel A showing the absence of glycosylation maturation of nicastrin after MNJ treatment. (C) Cell-based {gamma}-secretase assay. HEK cells stably expressing APP/Sw were transiently transfected with mNotch{Delta}E or NICD and treated with kifunensine (1 µg/ml), a mannosidase I inhibitor resulting in inhibition of mature glycosylation of nicastrin. Aß and NICD generation was analyzed as described. Note that inhibition of glycosylation of nicastrin does not inhibit the production of Aß nor NICD. (D) Inhibition of glycosylation has no effect on surface expression of nicastrin. HEK293 cells were treated with kifunensine (1 µg/ml) and surface biotinylated after 72 hours kifunensine treatment. Biotinylated proteins were precipitated using streptavidin beads and immunoblotted with B59.2 ({alpha}-nicastrin). Western blot analysis of total cell extracts is shown as a control. Nicastrin is labeled in both treated and untreated cells, demonstrating that immature glycosylated nicastrin can reach the cell surface under these conditions.

 

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